(1) Field of the Invention
The present invention relates to a novel use of a malaria antigen to immunise against malaria infection and disease. The invention relates in particular to the use of sporozoite antigens, in particular circumsporozoite (CS) protein or immunogenic fragments or derivatives thereof, combined with suitable adjuvants, to immunise malaria naïve individuals expecting to travel to endemic regions against malaria infection.
(2) Description of the Related Art
Malaria is one of the world's major health problems. During the 20th century, economic and social development, together with anti-malarial campaigns, have resulted in the eradication of malaria from large areas of the world, reducing the affected area of the earth's land surface from 50% to 27%. Nonetheless, given expected population growth it is projected that by 2010 half of the world's population, nearly 3.5 billion people, will be living in areas where malaria is transmitted (Hay, 2004). Current estimates suggest that there are well in excess of 1 million deaths due to malaria every year, and the economic costs for Africa alone are staggering (Bremen, 2004).
These figures highlight the global malaria crisis and the challenges it poses to the international health community. The reasons for this crisis are multiple and range from the emergence of widespread resistance to available, affordable and previously highly effective drugs, to the breakdown and inadequacy of health systems and the lack of resources. Unless ways are found to control this disease, global efforts to improve health and child survival, reduce poverty, increase security and strengthen the most vulnerable societies will fail.
Malaria also poses risks to those traveling to or working in endemic regions who normally live in malaria free countries. The risks may be greater to this traveler population because they do not have any background immunity to malaria from natural exposure. Another aspect of the risk incurred by a traveler to a malaria endemic region is that the disease is often mis-diagnosed in its early stages due to the flu-like symptoms. When the severity increases and malaria is finally diagnosed, it can be too late. Within a few days of the increased symptoms, death can result, for example, from cerebral malaria, or sometimes organ (e.g. liver or kidney) failure.
One of the most acute forms of the disease is caused by the protozoan parasite Plasmodium falciparum which is responsible for most of the mortality attributable to malaria. Another form of the disease is caused by Plasmodium vivax. P. vivax is the most widespread of all malarias. In addition to being present in tropical and sub-tropical regions, the ability of the parasite to complete its mosquito cycle at temperatures as low as 15 degrees Celsius, has allowed it to spread in temperate climates. However due to the fact that P. vivax infection is rarely fatal, the efforts to control P. vivax malaria (through vaccine development) are lagging behind those for P. falciparum. 
An observation made 30 years ago provides strong support for the belief that an effective malaria vaccine will eventually be developed. Mice and humans can be protected against malaria by immunisation with live, radiation-attenuated malaria sporozoites. The persistence of intra-hepatic stage in vivo is required to produce and maintain protective immunity, but the underlying mechanisms have not yet been completely defined. Antibodies, CD8 and CD4 T-cells (Hoffman, 1996) have been implicated as critical effector immune mediators.
The life cycle of Plasmodium sp (eg, P. falciparum or P. vivax) is complex, requiring two hosts, man and mosquito for completion. The infection of man is initiated by the inoculation of sporozoites in the saliva of an infected mosquito. The sporozoites migrate to the liver and there infect hepatocytes (liver stage) where they differentiate, via the exoerythrocytic intracellular stage, into the merozoite stage which infects red blood cells (RBC) to initiate cyclical replication in the asexual blood stage. The cycle is completed by the differentiation of a number of merozoites in the RBC into sexual stage gametocytes which are ingested by the mosquito, where they develop through a series of stages in the midgut to produce sporozoites which migrate to the salivary gland.
The sporozoite stage of Plasmodium sp (eg, P. falciparum or P. vivax) has been identified as one potential target of a malaria vaccine. The major surface protein of the sporozoite is known as circumsporozoite protein (CS protein). This protein has been cloned, expressed and sequenced for a variety of strains, for example for P. falciparum the NF54 strain, clone 3D7 (Caspers, 1989). The protein from strain 3D7 is characterised by having a central immunodominant repeat region comprising a tetrapeptide Asn-Ala-Asn-Pro (SEQ ID NO:1) repeated 40 times but interspersed with four minor repeats of the tetrapeptide Asn-Val-Asp-Pro (SEQ ID NO:2). In other strains the number of major and minor repeats varies as well as their relative position. This central portion is flanked by an N and C terminal portion composed of non-repetitive amino acid sequences designated as the repeatless portion of the CS protein.
GlaxoSmithKline Biologicals' RTS,S malaria vaccine based on CS protein has been under development since 1987 and is currently the most advanced malaria vaccine candidate being studied (Ballou, 2004). This vaccine specifically targets the pre-erythrocytic stage of P. falciparum. 
RTS,S/AS02A (RTS,S plus adjuvant AS02A which contains immunostimulants QS21, 3D-MPL and an oil in water emulsion) was used in consecutive Phase I studies undertaken in The Gambia involving adults (Doherty, 1999), children aged 6-11 and 1-5 years (Bojang, 2005), which confirmed that the vaccine was safe, well-tolerated and immunogenic. Subsequently a paediatric vaccine dose was selected and studied in a Phase I study involving Mozambican children aged 1-4 years where it was found to be safe, well tolerated and immunogenic (Macete).
The RTS,S/AS02A vaccine has also shown evidence of efficacy in clinical trials in the USA and in the field in West Africa. RTS,S/AS02A induces significant IgG antibody responses to P. falciparum circumsporozoite protein and substantial T-cell immunity (Lalvani, 1999; Sun, 2003). Efficacy against P. falciparum experimental challenge in malaria-naïve volunteers in the USA has been estimated to be about 30-50% on average (Stoute, 1997; Stoute 1998; Kester, 2001). The first of these studies (Stoute, 1997) was 86% effective in a small scale trial in which 6 out of 7 individuals immunized with RTS,S/AS02A were protected. Furthermore, a field study of semi-immune adults in The Gambia (preceeded by a safety study in Gambian adults (Doherty, 1999)) showed an overall efficacy of 34% over a period of one transmission season of 15 weeks, with 71% efficacy in the first nine weeks of follow-up and 0% efficacy thereafter (Bojang, 2001). These studies (Stoute, 1997; Stoute, 1998; Bojang, 2001; Kester, 2001) show efficacy against infection.
Results were recently reported from a trial using RTS,S/AS02A in young African children. It was discovered that the CS protein based RTS,S vaccine can confer not only protection against infection under natural exposure but also protection against a wide spectrum of clinical illness caused by P. falciparum. Children who received the RTS,S vaccine experienced fewer serious adverse events, hospitalisations, and severe complications from malaria, including death, than did those in the control group (Alonso, 2004).
Furthermore, the RTS,S vaccine efficacy against both new infections or clinical episodes appears either not to wane or to do so slowly. At the end of the 6 months follow up in the trial, the vaccine remained efficacious as there was a significant difference in the prevalence of infection. This is in contrast with previous trials in malaria naïve volunteers or Gambian adults which suggested that vaccine efficacy with RTS,S was short lived (Stoute, 1998; Bojang, 2001). Furthermore, after an additional follow-up period of 12 months, it was observed that the efficacy of the vaccine against an episode of clinical malaria did not significantly wane (Alonso, 2005).
Although the vaccine formulation described above shows clinical efficacy, additional improvements are still needed in order to increase both the number of individuals protected as well as the persistence of protection. New adjuvant formulations such as a formulation which contains QS21 and 3D-MPL in a liposome containing formulation (referred to herein as adjuvant B) have demonstrated a higher potency to boost T-cell immune response in various pre-clinical and clinical investigations.
In particular, what is needed for a vaccine for people who do not come from a malaria endemic region but are traveling for a limited period of time to regions where malaria is endemic, is better protection against infection. Clinical manifestation of malaria can only occur if there is a productive infection of the liver leading to the formation of merozoites and their release from the hepatic stage. These merezoites can then infect RBC and initiate the pathogenic blood stage of the parasite resulting in symptomatic clinical malaria. If there is no productive infection following exposure (i.e. no infection of the liver and/or no release of liver merozoites), then this is known as sterile immunity. A vaccine that would significantly reduce the risk of a productive liver infection, as defined above, following mosquito bites would be highly desirable for a traveler population that does not have pre-existing immunity, because by preventing the productive hepatic infection the vaccine would prevent any subsequent clinical manifestation. This can be contrasted with the aim of vaccine development targeting children or people in endemic regions, where the major aim would be to decrease the severity of disease and/or to decrease the number of episodes of disease, but not necessarily to prevent them completely. In theory, it would not be possible to indefinitely maintain sterile protection in people in endemic regions, and therefore they need to build up their own immunity by exposure to malaria infection. Furthermore, it may not be advisable to confer sterile protection on people living in endemic regions for an extended yet limited period of time.
We describe herein a challenge clinical trial consisting of a head to head comparison of RTS,S/AS02A versus RTS,S with a different adjuvant (adjuvant B) which contains QS21 and 3D-MPL in a formulation with cholesterol-containing liposomes, in a malaria naïve population (see Examples). Both T- and B-cell mediated immunity were investigated.
The results show that in a malaria naïve adult population the RTS,S antigen in combination with adjuvant B is greater than 50% more effective at protecting against productive hepatic infection following malaria challenge than RTS,S/AS02A. Thus, the RTS,S antigen in combination with adjuvant B is more effective in terms of the sterile protection which is required for malaria naïve individuals traveling to regions where malaria is endemic. This increased efficacy conferred by adjuvant B is associated with an increased antigen specific immune response (antibodies and CD4-Th1 T-cells).